Immunotherapeutic agent derived from bacteria and method for its manufacture

ABSTRACT

An immunotherapeutic agent is prepared from cells of E. coli or members of the genus Mycobacterium. The material is effective as an anti-tumor agent, an immunostimulant, and an adjuvant. Also disclosed is a method of evoking an immunostimulatory response through the activation of the RAS gene.

This application is a continuation of application Ser. No. 08/299,145,filed Aug. 31, 1994, now U.S. Pat. No. 5,527,770.

FIELD OF THE INVENTION

This invention relates generally to a bacterial cell preparation whichmanifests anti-tumor, adjuvant and immunostimulatory properties, and tomethods for the preparation of the material.

BACKGROUND OF THE INVENTION

The role of the immune system is critical in the control of diseasessuch as cancer, as well as diseases caused by external agents such asvirus or bacteria. Presently, there is great interest in the use oftherapeutic materials which can enhance the response of the immunesystem, particularly with regard to the treatment of cancer and AIDS. Ithas been known for some time that various bacteria manifest a stronganti-tumor and immunostimulatory effect. Also, it has been found thatthese bacteria can also act as an adjuvant material. An adjuvant is amaterial which, when introduced into an animal, along with an antigen,evokes and enhance production of antibodies to that antigen.

Various bacterial preparations have been investigated for use asimmunostimulatory agents. Freund's Complete Adjuvant (CFA) was developedin the early 1950's. It comprises a crude preparation of a bacteria ofthe genus Mycobacterium, particularly M. tuberculosis. CFA has beenfound to be a relatively potent adjuvant and has become a researchstandard; however, its use as a therapeutic agent, and in some instancesits use as a research material, has been limited by the fact that it isquite toxic. In an attempt to overcome the toxicity of CFA, variousother adjuvant materials have been developed; for example, as disclosedby Bennett et al. in "Journal of Immunological Methods" 153 (1992)31-40, a synthetic material comprising a water in oil emulsion ofsqualene together with a particular block copolymer has been found tohave adjuvant activity. This material is still somewhat toxic and it isemployed in a non-aqueous base and hence of limited utility.

Various bacterial preparations have been developed in an attempt toimprove upon CFA. As disclosed in U.S. Pat. No. 4,726,947, a relativelyhigh molecular weight extract of various species of Mycobacterium hasbeen found to have adjuvant and anti-tumor effects. As disclosed in U.S.Pat. No. 5,116,614, cell wall preparations of the bacterial genusNocardia have adjuvant and anti-tumor effects when coupled withparticular synthetic molecules.

All the prior art adjuvant and anti-tumor materials have been found tobe less than adequate for clinical applications. The immunostimulatoryeffect of prior art materials is generally far less than that of CFA.Furthermore, many of these materials are toxic and difficult to prepare.Furthermore, most are not aqueous based and, hence, their use is furthercomplicated. It will be appreciated that there is a need for animmunotherapeutic agent which is of high activity and low toxicity. Theagent should also be easy to prepare and administer. The presentinvention provides an immunotherapeutic agent derived from bacterialcells. The agent is highly active and of low toxicity. Its preparationis relatively simple and it is a stable, aqueous based material. Theseand other advantages of the present invention will be readily apparentfrom the discussion which follows:

BRIEF DESCRIPTION OF THE INVENTION

There is disclosed herein a method for preparing an immunotherapeuticagent. The method comprises the steps of culturing cells of a bacteriumselected from the group consisting of E. coli, and members of the genusMycobacterium. The cells are collected and their membranes disrupted, asfor example by ultrasonic energy. The disrupted cells are separated intosediment and supernatant liquid. The supernatant liquid is flocculatedso as to produce a solid material. The solid material is separated intoa first fraction having a molecular weight of more than 85,000 daltonsand a second fraction having a molecular weight of less than 85,000daltons. The second fraction contains the immunotherapeutic agent. Theagent is believed to include a material which has a molecular weight of919.2 daltons and comprises a glycopeptide, and may also include asecond material which has a molecular weight of approximately 65kilodaltons and comprises a glycosylated protein. It is believed thatthe therapeutic effects of the present invention are due to these twomaterials either taken singly or in combination.

There is also disclosed herein a method for simulating the immune systemof a cell. The method involves the activation of theRAS/RAF-1/MAP-Kinase pathway of the cell.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1C comprise a schematic depiction of one proposed mode by whichthe LMG of the present invention operates to activate the immune system.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an immunotherapeutic agent havinganti-tumor as well as adjuvant activity. The agent is derived frombacteria. The most preferred bacteria comprise members of the genusMycobacterium with one particularly preferred material being M.tuberculosis. Other members of the genus comprise M. Avium, M. Bovis andM. Smegmatis. It has also been found that the agent can be prepared fromE. coli. It is believed that the active material comprises aglycopeptide and/or a glycosylated protein. In the context of thepresent disclosure, the material of the present invention will bereferred to as "LMG (low molecular weight glycopeptide)." At present,the precise structure of the LMG has not been elucidated; however,parameters for its preparation are well defined. The active material mayinclude a first species of low molecular weight, i.e., approximately919.2 daltons as determined by mass spectroscopy. The basic structure ofthe first species as determined by 2-d nuclear magnetic resonance isthought to be: ##STR1## although it is to be understood that thestructure of the material prepared in accord with the invention mayactually be found to be different. The LMG may also include a secondactive species which has a molecular weight of about 65 kilodaltons andcomprises a glycosylated protein. Either of the species, or the two incombination, are responsible for the action of the LMG.

The LMG may be prepared from a number of Mycobacterium species with M.tuberculosis being one particularly preferred bacteria. It has alsosurprisingly been found that the LMG can be isolated from E coli.

The LMG was prepared from M. tuberculosis strain H37Ra as follows: thebacteria was grown in culture medium in accord with standard procedureswell known in the art. No particular restrictions are imposed on themethod of growing the microorganism and various media such as Sauton'smedium or glycerin-boullion medium may be employed. The bacteria arefiltered from the culture and dried to provide a powder. The powder issuspended in water and disrupted, for example, as by the input ofultrasonic energy. Care is taken that the sample be maintained at atemperature of 4° C. or less during the sonication process, so as tomaximize yield of LMG.

The emulsion resultant from the disruption is stirred for 48 hours,while being maintained at a temperature of 4° C. or less, after which itis centrifuged at 14000 rpm for 10 minutes, while being maintained at nomore than 4° C. The supernatant is collected and freeze dried, again ata temperature of no more than 4° C., to produce a dry material.

The freeze dried material is dissolved in water at an approximateconcentration of 20 milligrams per milliliter. The solution ismaintained at 4° C. and cold acetone is added dropwise with stirring.The total amount of acetone added is twice the volume of the water. Whenthe addition of acetone is completed, the vessel is tightly covered toprotect it from the atmosphere and the solution is stirred for 24 hours.Care is taken during the addition of acetone and the subsequent stirringto maintain the temperature at no more than 4° C. The solution resultantfrom these steps appears homogeneous.

The solution is centrifuged at 14,000 rpm for 10 minutes, at atemperature no more than 4° C. The supernatant solution is discarded andthe precipitated pellet is dissolved in water and freeze dried. The LMGis contained in this material.

The LMG is isolated by dissolving the freeze dried pellet material indistilled water, at an approximate concentration of 1milligram/milliliter. The dissolved material is then ultra filteredthrough a filter having a molecular weight cut-off point ofapproximately 85,000 daltons. The material which passes through thefilter comprises the LMG of the present invention.

In a variation of the above described process, the ultra filtration stepmay be replaced by high pressure liquid chromatography separation forthe isolation of the LMG.

The LMG has been found to have very good anti-tumor activity. BALB/Cmice were injected with B 1/6-F1 melanoma cells. After three injectionsof the LMG the primary tumor disappeared and no metastasis was detectedin the lung. In another experiment, NO/NO mice were injected with C-8161human melanoma, and after the tumor grew to approximately 4 millimeters,the mice were injected with LMG four times, at an interval of threedays, after which no tumors were detected in the lungs and the primarytumor at the injection site had disappeared. In another experiment,PC-3-1A human prostrate carcinoma cells were injected into nude mice.These cells typically will form multiple microscopic tumor colonies insuch mice after intravenous injection. In this experimental series, themice were injected with 20 milligrams of LMG in a PBS buffer 24 hoursafter the tumor cell line. The injection of the LMG was repeated fivedays later and no tumors were detected in the lungs or at the primaryinjection site.

The LMG has been found to be a potent adjuvant. It can be injected intoanimals by itself, typically in the form of a solution with PBS buffer,or together with an antigen. It has been found that the antibody titerresultant from the use of LMG is twice that of CFA, and no toxicreactions were detected. The typical amounts employed are 20 microgramsper 0.2 Ml.

The immunostimulatory effect of the material of the present invention isof significant utility in the treatment of AIDS. It has been found inanimal tests that LMG causes extensive proliferation of γS and αβ Tcells, an increase in the number of CD4 and T1 helper cells in the lymphnodes, an increase in interferon γ and a 30 fold increase in IL-2.

The immune system includes CD4 and CD8 cells in a generally fixed ratio.It is believed that when new cells are generated in the immune system,no distinction is made between the CD4 and CD8 cells. The AIDS virusonly attacks the CD4 cells, thereby decreasing their numbers. Inresponse to the decreasing level of CD4 cells, the body generates newCD4 cells, together with CD8 cells. The rise in CD8 cells serves tolimit the generation of further cells, including CD4 cells. Therefore,the CD4 level never rises fully and the strength of the immune systemultimately declines as a result of the homeostatic balance of the immunecells.

It has been found that the material of the present invention is animmunostimulant which can actually alter the ratio of CD4 to CD8 cells.In mice the normal ratio of CD8 to CD4 is in the range of 1:1.5 to 1:2.Eleven mice were injected with LMG. In 11 out of 11 mice it was foundthat the ratio of CD8:CD4 was raised to 1:4.

It has been determined, based upon experimental evidence, that the LMGof the present invention operates in a growth hormone-like manner tostimulate the immune system. This novel mode of action presents aheretofore unavailable biochemical pathway for the moderation of immunesystem response.

Referring now to FIGS. 1A-C, there is shown, in schematic form, theoperation of the present invention. FIG. 1A depicts a portion of a cellmembrane of a B cell. As is well known, the membrane 10 comprises abi-layer, and it includes a number of receptors 12 therein. Thereceptors are of different types, and are specific for variousmolecules. Associated with the cell, interiorally of the membrane 10, isa gene known as RAS. The RAS gene has previously been identified asbeing associated with cell growth and differentiation, and it canfunction as an oncogene. The RAS gene is in the GDP complex form, 14,all of said terms being well known in the art.

In the first step, as shown in FIG. 1B, the LMG of the present invention16, binds two particular receptors 12 on the cell membrane 10. The LMGactivates the receptors so as to provide a phosphate 18, typically inthe form of tyrosine phosphate. The activated receptors bind to theGRB2-SOS complex 20 which in turn activates the RAS gene, which toproduce the RAS-GTP complex 22.

As is shown in FIG. 1C, the RAS-GTP complex 22 activates RAS-1 kinase,24, possibly with help from a small number of other proteins. RAS-1kinase, 24, in turn, activates the MAP kinase cascade which activatesthe appropriate gene 36 which directs the release of transcriptionfactors and other cellular proteins that bring about the cell response.Specifically, RAS-1 kinase 24 activates the phosphorylation of MEK 26 toMEK-P 28; and MEK-P 28 activates the phosphorylation of MAP-K 32 toMAP-K-P 32. The phosphorylated MAP-K 32 acts within the cell nucleus 34to activate a specific gene 36. Activation occurs through cellularproteins and transcription factors JUN 38, FOS 40 and MYC 42, andpossibly others. Activation of the gene 36 brings about the release ofnovel proteins which lead to the proliferation of various lymphocytes.

The foregoing mechanism has been supported experimentally. It has beenfound that the LMG of the present invention is operative even when themajor histocompatibility complex MHC in B cells is blocked byactinomycin. It would normally be expected that blocking of MHC in the Bcells would inhibit T cell proliferation. Therefore, the activation ofthe B cells by the LMG must not proceed along the MHC pathway. It hasfurther been found that when human white blood cells are incubated forten minutes with the LMG of the present invention, the level of tyrosinephosphate increases, which is indicative of formation of the REC-Pcomplex. Also, it has been found that in the same cells, the level ofMAP-K-P increases.

Thus, it will be seen that the present invention provides a novelpathway whereby the RAS gene may be activated so as to activateleukocyte proliferation via a growth hormone-like pathway. Whileactivation is accomplished by the LMG of the present invention, it willbe appreciated that one of skill in the art could find other substanceswhich activate the pathway, and the extent of such activation could bemeasured in vitro by determining increases of the various materials suchas MAP-kinase, tyrosine phosphate, and the like along this pathway.

It will thus be appreciated that the present invention provides animmunotherapeutic agent comprising a low molecular weight glyco protein,which is of low toxicity and is highly effective as an anti-tumor agent,adjuvant and immune system stimulant. The agent of the present inventionhas significant utility as a research and therapeutic material.

It will be appreciated that the foregoing discussion and description ismerely illustrative of particular embodiments to the present invention,and is not meant to be a limitation upon the practice thereof. It is thefollowing claims, including all equivalents, which define the scope ofthe invention.

I claim:
 1. A method of preparing an immunotherapeutic agent comprisingthe steps of:culturing cells of a bacterium selected from the groupconsisting of E. coli, and members of the genus Mycobacterium;collecting the cells of said bacterium; disrupting the membranes of saidcells; separating said disrupted cells into sediment and supernatantliquid; flocculating the supernatant liquid; separating a solid materialfrom the flocculated supernatant liquid; and separating said solidmaterial into a first fraction having a molecular weight of about 85,000daltons and a second fraction having a molecular weight of less than85,000 daltons, wherein said immunotherapeutic agent comprises saidsecond fraction.
 2. A method as in claim 1, wherein the step ofculturing cells of a bacterium comprises culturing cells of M.tuberculosis.
 3. A method as in claim 1, wherein the step of disruptingthe membranes of said cells comprises suspending said cells in water andultrasonically disrupting their membranes.
 4. A method as in claim 1,wherein the step of separating said disrupted cells comprisescentrifuging said disrupted cells.
 5. A method as in claim 1, whereinthe step of flocculating the supernatant liquid comprises: freeze dryingthe supernatant to produce a dry material, dissolving the dry materialin water at a concentration of approximately 20 milligrams permilliliter, and adding cold acetone to said water in a ratio of 2 partsacetone to 1 part water.
 6. A method as in claim 1, wherein the step ofseparating a solid material from the flocculated supernatant liquidcomprises centrifuging said flocculated supernatant liquid.
 7. A methodas in claim 1, wherein the step of separating said solid materialcomprises dissolving said solid material in water and passing saiddissolved solid material through an ultrafilter.
 8. An immunotherapeuticagent obtained from bacteria by a process comprising:culturing cells ofa bacterium selected from the group consisting of members of the genusMycobacterium, and E. coli; collecting the cells of said bacterium;disrupting the membranes of said cells; separating said disrupted cellsinto sediment and supernatant liquid; flocculating the supernatantliquid; separating a solid material from the flocculated supernatantliquid; and separating said solid material into a first fraction havinga molecular weight of about 85,000 daltons and a second fraction havinga molecular weight of less than 85,000 daltons, wherein saidimmunotherapeutic agent is present in said second fraction.
 9. Animmunotherapeutic agent as in claim 8, which incudes a low molecularweight glycopeptide which has a molecular weight of 919.2 daltons and abasic structure represented by the formula: ##STR2##
 10. Animmunotherapeutic agent as in claim 8, which includes a glycosylatedprotein having a molecular weight of approximately 65,000 daltons.
 11. Amethod for stimulating the immune system of a cell comprising activatingthe intercellular RAS/RAF-1/MAP-kinase pathway of said cell.
 12. Amethod as in claim 11, wherein the step of activating saidRAS/RAF-1/MAP-kinase pathway comprises exposing said cell to animmunotherapeutic agent obtained from bacteria by a processcomprising:culturing cells of a bacterium selected from the groupconsisting of members of the genus Mycobacterium, and E. coli;collecting the cells of said bacterium; disrupting the membranes of saidcells; separating said disrupted cells into sediment and supernatantliquid; flocculating the supernatant liquid; separating a solid materialfrom the flocculated supernatant liquid; and separating said solidmaterial into a first fraction having a molecular weight of about 85,000daltons and a second fraction having a molecular weight of less than85,000 daltons, wherein said immunotherapeutic agent is present in saidsecond fraction.